The primary goal of our work is to understand how exogenous water- soluble proteins attain a functional intracellular or membrane-embedded state. The proteins of interest are diphtheria toxin (DTx), Pseudomonas toxin (PTx), tumor necrosis factor (TNF) and lymphotoxin (LT). While acidification is an obligatory trigger of the entry mechanisms of DTx and PTx, our comparative studies show that a 15-min exposure to pH 5.3 enhances the cytotoxic activities of all four proteins. Decreasing pH also promotes conformational changes that are in concert with increased lipid vesicle binding and insertion activities. We propose to continue previously defined projects that focus on the relationships between protein structure and membrane interaction, between membrane interaction and attainment of functional states. Photoreactive probes and other protein-tagging procedures will be used to monitor events within the membrane bilayer, including the kinetics and directionality of protein penetration and the fate of membrane-inserted peptides. New probes have been synthesized to expedite identification of photolabeled domains. Vesicles and simple biological membranes will be used as targets; a model "endosome" vesicle system that we designed will enable us to monitor protein traversal to the trans surface and the factors involved (pH, reduction potential, electrochemical gradient, etc.). Our recent discoveries that DTx and PTx possess intrinsic nuclease activity and that TNF and LT form ion-channels and lead to Na+ uptake in target cells warrant an in-depth characterization of the mechanisms involved. Our hypothesis is that these activities have direct roles in target cell destruction. Interestingly, all four proteins induce chromosomal degradation, membrane blebbing, and ultimately cell lysis. Although all cells have TNF/LT receptors, only malignant cells are killed. Moreover, the translation inhibition activities of DTx and PTx do not correlate with DNA degradation or cell lysis. We propose to exploit 1) unique temperature-sensitive mutant forms of the A domain of DTx to unravel the intracellular role of nuclease activity, 2) ion- sensitive dyes and patch clamp methods to establish the relationship between TNF/LT-induced Na+ influx, channel activity, and cell responsiveness, 3) anti-receptor antibodies, liposome-embedded TNF, and receptor-bearing and deficient cell lines, vesicles and planar membranes to clarify receptor function, and 4) treatments that are known to inhibit the TNF channel (e.g., Ca2+) or inhibit TNF-mediated cytolysis (e.g., certain amiloride analogues) to examine the physiological significance of TNF channels. Relevant ligand-protein interactions (e.g., DNA-DTx binding) will be characterized. Knowledge gained from these studies should lead to: more effective use (and possibly new forms) of TNF/LT for human cancer treatment, more effective "antidotes" for the debilitating and often lethal effects of TNF/LT (e.g., septic shock syndrome, cachexia), and more effective "magic bullets" and toxin gene constructs for use in basic and medical research.